摘要
背景:三花接骨散为临床骨折筋伤常用药,使用效果良好,但缺少相关实验学依据,其作用机制尚不明确。目的:观察三花接骨散对成骨细胞Wnt/β-Catenin信号通路的影响。方法:首先预实验筛选三花接骨散干预成骨细胞的最佳浓度,之后采用CCK-8法进行成骨细胞增殖实验,碱性磷酸酶检测成骨细胞成熟度,RT-PCR检测碱性磷酸酶、Runt相关转录因子2、骨钙蛋白、骨桥蛋白、胶原酶Ⅰ、Axin2、Dkk4、Lef-1、Tcf-1 mRNA表达,Western blot法检测β-Catenin、P-β-Catenin蛋白表达。结果与结论:(1)成骨细胞培养5 d后,10 mg/L质量浓度时成骨细胞数量最多;(2)成骨细胞培养21 d后,三花接骨散不同浓度组的细胞增殖数量均有增加,以10 mg/L组明显,但20 mg/L已有减少的趋势;(3)成骨细胞培养7 d后,三花接骨散低、中、高剂量组碱性磷酸酶表达均有增加,以高剂量组(20 mg/L)明显;(4)成骨细胞培养7 d后,三花接骨散低、中、高剂量组碱性磷酸酶、Runt相关转录因子2、骨钙蛋白,骨桥蛋白,胶原酶ⅠmRNA表达均有增加,以高剂量组(20 mg/L)明显;(5)成骨细胞培养21 d后,三花接骨散低、中、高剂量组Axin2、Dkk4 mRNA表达均有降低,Lef-1、Tcf-1 mRNA表达增加,以高剂量组(20 mg/L)明显;(6)成骨细胞培养21 d后,三花接骨散低、中、高剂量组P-β-Catenin蛋白表达均有降低,β-Catenin蛋白表达增加,均以高剂量组(20 mg/L)明显;(7)提示三花接骨散可调控成骨细胞Wnt/β-Catenin信号通路,促进碱性磷酸酶、Runt相关转录因子2、骨钙蛋白、骨桥蛋白、胶原酶Ⅰ、Lef-1、Tcf-1 mRNA以及β-Catenin蛋白表达,降低Axin2、Dkk4 mRNA以及P-β-Catenin蛋白表达,促进成骨细胞的增殖,这可能是其促进骨折愈合的作用机制。
BACKGROUND:Sanhua Jiegu San is a common drug for clinical fracture and tendon injury,which have good effect.However,there is a lack of relevant experimental evidence,and its mechanism of action remains unclear.OBJECTIVE:To observe the effect of Sanhua Jiegu San on Wnt/β-catenin signaling pathway in osteoblasts.METHODS:Firstly,the optimal concentration of Sanhua Jiegu San for osteoblast intervention was screened by pre-experiment.Then,the proliferation of osteoblasts was tested by cell counting kit-8 method.The maturity of osteoblasts was detected by alkaline phosphatase assay.The mRNA expressions of alkaline phosphatase,Runt-related transcription factor 2,osteocalcin,osteopontin,collagenaseⅠ,Axin2,Dkk4,Lef-1,and Tcf-1 were detected by PCR.The protein expressions ofβ-Catenin and P-β-Catenin were detected by western blot method.RESULTS AND CONCLUSION:After 5 days of osteoblast culture,the number of osteoblasts reached the maximum at the concentration of 10 mg/L.After 21 days of osteoblast culture,the proliferation of cells in different concentration groups of Sanhua Jiegu San increased,which was obvious in the 10 mg/L group,but tended to decrease at 20 mg/L.After 7 days of osteoblast culture,alkaline phosphatase expression was increased in the low,medium and high concentration groups,especially in the high concentration group(20 mg/L).After 7 days of osteoblast culture,mRNA expressions of alkaline phosphatase,Runt-related transcription factor 2,osteocalcin,osteopontin,collagenaseⅠwere increased in the low,medium and high concentration groups,especially in the high concentration group(20 mg/L).After 21 days of osteoblast culture,the mRNA expressions of Axin2 and Dkk4 were decreased in the low,medium and high concentration groups,while the mRNA expressions of Lef-1 and Tcf-1 were increased,especially in the high concentration group(20 mg/L).After 21 days of osteoblast culture,the expression of P-β-Catenin protein was decreased in the low,medium and high concentration groups,while the expression ofβ-Catenin p
作者
段嘉豪
谭旭仪
卢敏
邝高艳
方传龙
肖满
Duan Jiahao;Tan Xuyi;Lu Min;Kuang Gaoyan;Fang Chuanlong;Xiao Man(Hunan University of Chinese Medicine,Changsha 410208,Hunan Province,China;Department of Orthopedics,Affiliated Hospital of Hunan Academy of Chinese Medical Science,Changsha 410006,Hunan Province,China;Department of Orthopedics,The First Hospital of Hunan University of Chinese Medicine,Changsha 410021,Hunan Province,China;Hunan Fangsheng Pharmaceutical Co.,Ltd.,Changsha 410205,Hunan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第20期3230-3235,共6页
Chinese Journal of Tissue Engineering Research
基金
湖南中医药大学横向课题:三花接骨散促骨形成细胞/分子生物学机制研究。
